Human corneal fibrosis and scarring is a usual outcome following corneal injury. Despite significant advancements in the field, current treatments are limited, the visual recovery is poor. One of the problems is that corneal wound healing is a complex process, involving corneal cells, extracellular matrix (ECM) components and growth factors. The mechanism by which corneal fibrosis can be prevented is still unknown. Further understanding is required, along with new treatments to treat corneal scarring. The depth of the problem is clear when we consider that a total of 250 million people worldwide have compromised vision and around 6 million have been blinded, majorly due to corneal fibrosis and scarring. The main characteristic of a corneal scar is the presence of myofibroblasts, often indicated by the expression of ?-SMA (smooth muscle actin), and the excessive and improper deposition of ECM components such as type III collagen. Preventing scar formation would be ideal; however, studies investigating the development of non-fibrotic healing in human corneas are extremely limited. There are two main reasons for that: 1) Unavailability of human tissue makes investigations difficult, and 2) Mechanistic pathways of corneal scarring are not fully understood. We propose to investigate a novel player in corneal fibrosis known as sphingolipids. Sphingolipids have been recently linked to fibrosis in a variety of tissues and they are found to be in close connection to the TGF- (transforming growth factor) pathway, a cascade of events initiated by TGF- isoforms after binding to its receptors on cell surface, which has been shown to be involved in corneal fibrotic development. We will utilize our previous experience using our human 3D culture model in order to investigate the effect of sphingolipids in human corneal fibrosis. Preliminary data shows a direct link between sphingolipids and TGF- pathways. This would be a unique tool to understand the mechanism by which sphingolipids act on corneal fibrosis. We also have the corresponding mouse models in order to correlate our in vitro findings to in vivo. We propose two specific aims to examine the following questions: First, how are sphingolipids, specifically sphingosine-1-phosphate (S1P), and their receptors related to TGF- and how they alter the fibrotic cascade in vitro? Second, can we use genetic knockout mouse lines of S1P biosynthesis to determine the effect of sphingolipids in vivo? Relevance to Public Health - Corneal scarring can lead to complete or partial loss of vision. Investigation of a novel mechanism and pathway that can regulate non-fibrotic corneal development will provide important solutions to this sight threatening process. Ultimately this study will lead to the development of therapeutic agents that can treat corneal scarring in humans.